Abstract
Heterologous expression is an efficient strategy for target protein production. Dlt operon plays the important role in the d-alanylation of lipoteichoic acid, which might affect the net negative charge of cell wall for protein secretion. In this study, dlt operon was deleted to improve the target protein production, and nattokinase, α-amylase and β-mannanase with different isoelectric points (PIs) were served as the target proteins. Firstly, our results implied that deletions of dltA, dltB, dltC and dltD improved the net negative charge of cell wall for extracellular protein secretion respectively, and among which, the dltB deficient strain DW2ΔdltB showed the best performance, its nattokinase (PI: 8.60) activity was increased by 27.50% compared with that of DW2/pP43SacCNK. Then, the dltABCD mutant strain was constructed, and the net negative charge and nattokinase activity were increased by 55.57% and 37.13% respectively, due to the deficiency of dltABCD. Moreover, it was confirmed that the activities of α-amylase (PI: 6.26) and β-mannanase (PI: 5.75) were enhanced by 44.53% and 53.06% in the dltABCD deficient strains, respectively. Collectively, this study provided a strategy that deletion of dlt operon improves the protein secretion in B. licheniformis, and which strategy was more conducive to the target protein with lower PI.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cai D, Wei X, Qiu Y, Chen Y, Chen J, Wen Z, Chen S (2016) High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase. J Appl Microbiol 121(3):704–712
Cai D, Wang H, He P, Zhu C, Wang Q, Wei X, Nomura CT, Chen S (2017) A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis. Microb Cell Fact 16(1):70
Cai D, Hu S, Chen Y, Liu L, Yang S, Ma X, Chen S (2018) Enhanced production of poly-gamma-glutamic acid by overexpression of the global anaerobic regulator Fnr in Bacillus licheniformis WX-02. Appl Biochem Biotechnol. https://doi.org/10.1007/s12010-018-2693-7
Cao H, van Heel AJ, Ahmed H, Mols M, Kuipers OP (2017) Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed alpha-amylases. Microb Cell Fact 16(1):56
Chen J, Fu G, Gai Y, Zheng P, Zhang D, Wen J (2015) Combinatorial sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression. Microb Cell Fact 14:92
Craynest M, Jorgensen S, Sarvas M, Kontinen VP (2003) Enhanced secretion of heterologous cyclodextrin glycosyltransferase by a mutant of Bacillus licheniformis defective in the D-alanylation of teichoic acids. Lett Appl Microbiol 37(1):75–80
Degering C, Eggert T, Puls M, Bongaerts J, Evers S, Maurer KH, Jaeger KE (2010) Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and heterologous signal peptides. Appl Environ Microbiol 76(19):6370–6376
Harwood CR, Cranenburgh R (2008) Bacillus protein secretion: an unfolding story. Trend Microbiol 16(2):73–79
He P, Zhang Z, Cai D, Chen Y, Wang H, Wei X, Li S, Chen S (2017) High-level production of alpha-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis. Biotechnol Lett 39(9):1389–1394
Hyyrylainen HL, Vitikainen M, Thwaite J, Wu H, Sarvas M, Harwood CR, Kontinen VP, Stephenson K (2000) D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis. J Biol Chem 275(35):26696–26703
Hyyrylainen HL, Pietiainen M, Lunden T, Ekman A, Gardemeister M, Murtomaki-Repo S, Antelmann H, Hecker M, Valmu L, Sarvas M, Kontinen VP (2007) The density of negative charge in the cell wall influences two-component signal transduction in Bacillus subtilis. Microbiology 153(Pt 7):2126–2136
Kang Z, Yang S, Du G, Chen J (2014) Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species. J Ind Microbiol Biotechnol 41(11):1599–1607
Kiriukhin MY, Neuhaus FC (2001) D-alanylation of lipoteichoic acid: role of the D-alanyl carrier protein in acylation. J Bacteriol 183(6):2051–2058
Kovacs M, Halfmann A, Fedtke I, Heintz M, Peschel A, Vollmer W, Hakenbeck R, Bruckner R (2006) A functional dlt operon, encoding proteins required for incorporation of d-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae. J Bacteriol 188(16):5797–5805
Lopez CS, Alice AF, Heras H, Rivas EA, Sanchez-Rivas C (2006) Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity. Microbiology 152(Pt 3):605–616
Saar-Dover R, Bitler A, Nezer R, Shmuel-Galia L, Firon A, Shimoni E, Trieu-Cuot P, Shai Y (2012) D-Alanylation of lipoteichoic acids confers resistance to cationic peptides in group B Streptococcus by increasing the cell wall density. PLoS Pathog 8(9):e1002891
Saeui CT, Mathew MP, Liu L, Urias E, Yarema KJ (2015) Cell surface and membrane engineering: emerging technologies and applications. J Funct Biomater 6(2):454–485
Sherkhanov S, Korman TP, Bowie JU (2014) Improving the tolerance of Escherichia coli to medium-chain fatty acid production. Metab Eng 25:1–7
Song Y, Fu G, Dong H, Li J, Du Y, Zhang D (2017) High-Efficiency secretion of beta-mannanase in Bacillus subtilis through protein synthesis and secretion optimization. J Agric Food Chem 65(12):2540–2548
Spatafora GA, Sheets M, June R, Luyimbazi D, Howard K, Hulbert R, Barnard D, el Janne M, Hudson MC (1999) Regulated expression of the Streptococcus mutans dlt genes correlates with intracellular polysaccharide accumulation. J Bacteriol 181(8):2363–2372
Stephenson K, Jensen CL, Jorgensen ST, Lakey JH, Harwood CR (2000) The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis. Biochem J 350 (Pt 1):31–9
Su L, Jiang Q, Yu L, Wu J (2017) Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C. Microb Cell Fact 16(1):24
Tan Z, Khakbaz P, Chen Y, Lombardo J, Yoon JM, Shanks JV, Klauda JB, Jarboe LR (2017) Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables. Metab Eng 44:1–12
Thwaite JE, Baillie LW, Carter NM, Stephenson K, Rees M, Harwood CR, Emmerson PT (2002) Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis. Appl Environ Microbiol 68(1):227–234
Ueda M (2016) Establishment of cell surface engineering and its development. Biosci Biotechnol Biochem 80(7):1243–1253
van Dijl JM, Hecker M (2013) Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb Cell Fact 12:3
Vitikainen M, Hyyrylainen HL, Kivimaki A, Kontinen VP, Sarvas M (2005) Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation. J Appl Microbiol 99(2):363–375
Wang D, Wang Q, Qiu Y, Nomura CT, Li J, Chen S (2017a) Untangling the transcription regulatory network of the bacitracin synthase operon in Bacillus licheniformis DW2. Res Microbiol 168(6):515–523
Wang Q, Zheng H, Wan X, Huang H, Li J, Nomura CT, Wang C, Chen S (2017b) Optimization of inexpensive agricultural by-products as raw materials for bacitracin production in Bacillus licheniformis DW2. Appl Biochem Biotechnol 183(4):1146–1157
Wecke J, Perego M, Fischer W (1996) D-Alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity. Microb Drug Resist 2(1):123–129
Wei X, Zhou Y, Chen J, Cai D, Wang D, Qi G, Chen S (2015) Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization. J Ind Microbiol Biotechnol 42(2):287–295
Wu T, Ye L, Zhao D, Li S, Li Q, Zhang B, Bi C, Zhang X (2017) Membrane engineering—A novel strategy to enhance the production and accumulation of beta-carotene in Escherichia coli. Metab Eng 43(Pt A):85–91
Yang S, Du G, Chen J, Kang Z (2017) Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Appl Microbiol Biotechnol 101(10):4151–4161
Zhang J, Kang Z, Ling Z, Cao W, Liu L, Wang M, Du G, Chen J (2013) High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements. Bioresour Technol 146:543–548
Acknowledgements
This work was supported by the National Program on Key Basic Research Project (973 Program, No. 2015CB150505), the Technical Innovation Special Fund of Hubei Province (2018ACA149), the Science and Technology Program of Wuhan (20160201010086).
Author information
Authors and Affiliations
Contributions
DC and SC designed the study. YC, DC and PH carried out the molecular biology studies and construction of engineering strains. YC, FM and QZ carried out the fermentation studies. YC, DC, XM and SC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Chen, Y., Cai, D., He, P. et al. Enhanced production of heterologous proteins by Bacillus licheniformis with defective d-alanylation of lipoteichoic acid. World J Microbiol Biotechnol 34, 135 (2018). https://doi.org/10.1007/s11274-018-2520-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-018-2520-x